JP2005031086A - Castable diffusion membrane for enzyme-based sensor application - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a membrane having good processability and a defined permeability, usable in a plurality of measurements. <P>SOLUTION: A diffusion membrane for use in an enzyme-based sensor, a sensor having the diffusion membrane, and the use of the enzyme-based sensor for the detection and/or determination of a substance, particularly an enzyme substrate (e.g., glucose), are disclosed. The membrane contains at least one kind of polymer material and pore maker particles dispersed in the at least one kind of polymer material. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a diffusion membrane used in an enzyme-based sensor, a sensor provided with a diffusion membrane, a method for producing an enzyme-based sensor, and a method for detecting and / or quantifying a substance, particularly an enzyme substrate (for example, glucose). Relates to the use of enzyme-based sensors.

  Enzyme-based sensors are widely used to quantitatively and qualitatively measure target substances in blood and other body fluids. Enzyme based sensors are used in particular to measure enzyme substrates. An enzyme-based sensor performs a so-called sensing reaction (sometimes referred to in the art as a “transducer reaction”). In this reaction, the substance is converted into another substance that can be detected directly or indirectly with the involvement of at least one enzyme. An example of such a sensing reaction is enzyme-catalyzed oxidation of glucose. Usually, this reaction uses oxygen as the electron acceptor. In the course of the reaction, glucose is converted to gluconolactone and oxygen is converted to hydrogen peroxide. The sensing reaction can measure either glucose and oxygen consumption or hydrogen peroxide or gluconolactone production.

  Enzyme-based sensors usually comprise several layers, in particular an enzyme layer and a cover membrane or outer layer. This cover membrane is in direct contact with the sample and limits the diffusion of substances required for the sensing reaction, in particular enzyme substrates or co-substrates.

  Enzyme based sensors can be provided as electrochemical sensors or as light sensors (optodes). About the structure and function of glucose optode, it describes in patent document 1, for example. About the structure and function of an electrochemical glucose sensor, it describes in patent document 2, for example.

  In particular, enzyme-based sensors used to measure glucose, lactate, or creatinine are preferably constructed using oxidoreductase, and detection is based on oxygen consumption. In this case, the requirement is placed on the sensor that the cover membrane must be a porous polymer membrane or at least a permeable polymer membrane that controls the permeation of both the enzyme substrate and oxygen.

  Currently available diffusion membranes for use in enzyme-based sensors have various drawbacks. According to one method known in the art, a cover membrane for use in an enzyme-based sensor is preformed consisting of a microporous structure obtained from non-hydratable polymers such as polycarbonate, polypropylene, and polyester. Film. The porosity of such a film is provided by physical means, for example by neutron or argon track etching. Glucose and oxygen pass through such membranes primarily through these pores filled with blood or other body fluids. One major drawback is that such membranes are preformed and not castable. A preformed membrane must be attached to the enzyme layer. In most cases, the membrane is mechanically attached to the enzyme layer. Such mechanical attachment is expensive and technically complex. A further problem arises unless it becomes easy to apply the + membrane to the underlying layer without generating bubbles. For example, a similar problem occurs when a film is bonded to the lower layer with an adhesive.

  In other methods known in the art, the cover membrane is castable. Such a cover film is generally formed by applying a polymer solution to the enzyme layer and evaporating the solvent. Such a membrane consists of a polymer structure having a hydrophilic region and a hydrophobic region. When exposed to water, the hydrophilic region of the membrane absorbs water and thus takes a swollen structure to provide, for example, a permeation pathway for glucose. However, these membranes do not provide a clear porosity. In this method, not all polymers are suitable because the polymer itself must provide permeability, so there is a limit to the choice of polymer.

  One drawback is that polymers that are suitable for use in castable membranes are often only soluble in irritating or toxic solvents. Examples of this include cellulose acetate, which is soluble in DMSO and acetone, and PVC, which is soluble in tetrahydrofuran and cyclohexanone. These situations are associated not only with safety reasons, but also with the possibility that enzymes present in the enzyme layer can be destroyed by these solvents. Furthermore, the effectiveness of such membranes depends on the dispersion of hydrophilic domains within the hydrophobic matrix. It is difficult to achieve uniform dispersion throughout the membrane manufacturing process, resulting in a non-uniform distribution of hydrophilic domains. For this reason, for example, reproducibility becomes insufficient.

Offenbacher et al. (Patent Document 3) describe a cover membrane having better coating reproducibility. The membrane is made of a PVC copolymer that can be very satisfactorily adjusted for permeability by the presence of a hydrophilic copolymer component. However, the regeneration of the oxygen reservoir of the sensor is so slow that such PVC cover membranes have limitations in multiple measurements when used for sensors based on oxygen consumption.
U.S. Patent 6,107,083 WO99 / 30152 pamphlet U.S. Patent No. 6,214,185

  The object of the present invention is to at least partially overcome the above-mentioned drawbacks associated with membranes for use in enzyme-based sensors. In particular, an object is to provide a castable film having good processability. A further object is to provide a membrane with a clear permeability that can be used for multiple measurements.

  Accordingly, the present invention relates to a diffusion membrane for use in an enzyme based sensor. The membrane includes (a) at least one polymeric material and (b) pore maker particles dispersed in the at least one polymeric material.

  The membrane according to the present invention comprises pore-forming particles dispersed in one or more polymer materials. The porosity of the membrane is provided by the pore-forming particles. Therefore, there is no restriction in selecting a polymer from the viewpoint of permeability or porosity.

  The polymeric material used in the diffusion membranes of the present invention may generally be any castable polymeric material or mixture of polymeric materials. For example, a non-toxic material or an easily applicable material can be used in accordance with the intended use.

  Preferred polymeric materials are selected from the group consisting of water insoluble polymers, preferably selected from polyurethanes, polyacrylamides, polystyrenes, polyvinyl esters, and copolymers (eg, copolymers of butadiene and styrene).

  Since there are various possibilities for the polymer material, it is possible to easily provide a castable diffusion film that can be directly coated with reproducibility. The diffusion film can be coated, for example, on the lower layer, preferably on the enzyme layer. It is an advantage of the diffusion membranes of the present invention that coating the diffusion membrane directly on the enzyme layer does not change the associated enzyme activity. Furthermore, it is possible to easily provide a diffusion film that is insoluble in body fluids such as blood.

  The diffusion film according to the present invention includes pore-forming particles dispersed in a layer-forming polymer material. The pore-forming particles provide membrane porosity. The particles used as pore-makers are preferably stable particles or a mixture of such particles with an inherently distinct porosity. The size of the pore-forming particles is preferably about 0.5-100 μm, in particular about 1-50 μm.

  The membrane has a certain porosity provided by the pores formed by the pore-forming particles according to the present invention. Depending on the application of the membrane, the pore size can be varied. The size of the pores is preferably in the range of the size of the pore-forming particles.

  The diffusion membrane according to the present invention is preferably finely divided in an amount of about 0.5 to 70 wt%, more preferably about 0.7 to 50 wt%, and most preferably about 1 to 40 wt%, based on the total weight of the dry membrane. Contains pore-forming particles. If the proportion of pore-forming particles exceeds a certain limit, the membrane becomes mechanically unstable, and if the amount of pore-forming particles added is too small, the membrane may become impermeable.

  For use as pore-forming particles in a membrane, essentially all stable particles and mixtures of such particles having an inherently distinct porosity are useful. Depending on the desired application and / or pore size, suitable particles can be selected. Examples of suitable pore-forming particles include inorganic or organic particles made from Kieselguhr, silica gel, cellulose, precipitated gypsum, kaolin, glass, and the like. Particularly preferred particles are particles based on silicon dioxide, in particular particles based on diatomaceous earth. A particularly preferred diatomaceous earth is that sold under the trade name Celatom®.

  The diffusion film according to the present invention further includes other components such as carbon black for blocking light, pigments such as titanium dioxide for improving the film's remission characteristics, or wetting agents. May contain.

  The thickness of the diffusion film according to the present invention can be selected flexibly according to the desired application and / or permeation rate. Suitable thicknesses are in the range of about 0.5 to 1000 μm, preferably about 3 to 500 μm, most preferably 5 to 100 μm.

  Thus, the permeation of the diffusion membrane can be easily adjusted by changing the thickness of the coating and / or the concentration of pore-forming particles.

  In one embodiment of the diffusion membrane according to the present invention, the size of the pore-forming particles corresponds at least to the layer thickness of the diffusion membrane, and the relationship between the particle size and the layer thickness is about 1: 1 or ≧ 1. : 1. In this embodiment, the size of the pore-forming particles is selected such that a single particle or cluster of single particles forms a continuous pore from the sensor surface to the enzyme layer. Therefore, diffusion of the material is brought about.

  A further aspect of the present invention is an enzyme based sensor comprising a diffusion membrane as described above. The sensor according to the present invention comprises several layers, at least one of which is an enzyme layer. The enzyme-based sensor according to the present invention further comprises a cover layer and at least one lower layer. Depending on the type of sensor, the further layer may be, for example, an interference prevention layer, a light blocking layer, a conductive layer, an indicator layer, or a base electrode.

  Since the permeability of the diffusion film can be adjusted as desired, the sensor can be rapidly regenerated by using this diffusion film. For example, in the case of a sensing reaction based on oxygen consumption, the permeation of oxygen can be adjusted so that the regeneration of the sensor (eg, regeneration of the oxygen reservoir) is very fast. Thus, the sensor of the present invention can also be used for multiple measurements.

  The enzyme layer of the enzyme-based sensor may contain an oxidase such as, for example, glucose oxidase, cholesterol oxidase, or lactate oxidase. The enzyme layer may also contain an enzyme mixture such as an enzyme cascade that allows detection of an analyte that cannot be directly detected, such as creatine. Creatine cannot be oxidized enzymatically by simple enzymes, but with several enzymatic steps, an analyte derivative that can be detected by optical or amperometric means can be produced. Suitable enzyme cascade systems for the detection and / or quantification of creatinine include, for example, creatinine amide hydrolase, creatinine amide hydrolase, and sarcosine oxidase.

  In the sensor according to the present invention, the diffusion film is preferably deposited as a cover layer. In this case, a stable cover layer is formed after solvent evaporation of the dispersion. The diffusion membrane is more preferably coated directly on the lower layer, preferably on the enzyme layer. By coating the membrane directly, the membrane layer is advantageously attached to the lower layer by physical adhesion without mechanical fixation and / or the use of an adhesive.

  When a diffusion membrane is used as the cover layer, the diffusion membrane is in direct contact with the test sample and regulates the diffusion of a substance (preferably a substrate or co-substrate) required for the sensing reaction. Therefore, a sensor having controlled permeability is provided by the diffusion film according to the present invention.

  The enzyme-based sensor according to the present invention can basically constitute any kind of biosensor. An example of a suitable biosensor is, for example, an optical sensor. In a preferred light sensor, consumption of oxygen based on an enzymatic reaction can be detected using an appropriate dye that is sensitive to oxygen (eg, a luminescent dye that can be quenched by oxygen). Furthermore, electrochemical sensors are also suitable for use with the present invention.

  In particular, if the membrane according to the present invention is used by being attached to a sensor that uses oxygen consumption as an analyte quantification means, a great advantage can be obtained. The pores formed by the pore-forming particles allow for the modulation of the diffusion of analyte molecules (eg glucose) through the membrane, and the choice of polymer affects the oxygen permeability. Oxygen permeation through the membrane can be effected to some extent through the pores formed by the pore-forming particles, but in particular if the polymer layer has a high oxygen permeability, it is essentially affected by the polymer layer. receive.

  A further aspect of the invention is the use of the enzyme-based sensor described above for detecting or quantifying a substance (preferably an enzyme substrate).

  In the medical field, for example, it can be used to measure physiological parameters. Measurement and / or detection can be performed in any fluid, for example, various body fluids such as blood, serum, plasma, urine and the like. A preferred use of the sensor is the detection and / or measurement of an analyte in blood.

  Possible uses of the sensor according to the invention include, for example, the determination of blood glucose in patients suffering from diabetes. Other metabolites that can be quantified using the enzyme-based sensor according to the invention are, for example, cholesterol or urea.

  Another possible use of the sensor according to the invention is in the field of environmental analysis, process control in biotechnology and food control.

  An enzyme-based sensor can be used according to the present invention to quantify and / or detect a wide variety of substances such as enzyme substrates and / or co-substrates. Suitable enzyme substrates are, for example, cholesterol, sucrose, glutamate, ethanol, ascorbic acid, fructose, pyruvate, ammonium, nitrite, nitrate, phenol, NADH, glucose, lactate, or creatinine. Preferably, glucose, lactate, or creatinine is quantified and / or detected. A particularly preferred substance to be detected and / or quantified is glucose.

  Since regeneration of enzyme-based sensors can be affected by regulating permeation, regeneration is fast enough to make multiple measurements. A preferred use of the sensor is to make multiple measurements. In addition, enzyme-based sensors can be utilized in any sensor application known in the art. For example, it can be used as a sensor for single use or a durability sensor for multiple use.

A further subject matter of the present invention is a method for the production of a sensor based on the enzymes described above. This method
(i) (a) at least one polymer material, and
(b) forming a dispersion containing pore-forming particles;
(ii) casting the dispersion directly onto the lower layer to form a diffusion film;
(iii) optionally, drying the dispersion;
including.

  According to the method according to the invention, a wide range of polymer materials can be selected, so that the membrane can be cast directly. If desired, it is also possible to work without solvents by using suitable polymer materials. Furthermore, the materials can be selected so that the dispersion does not need to be heated. Therefore, handling is facilitated by using the method according to the present invention.

  According to this method, the membrane can be applied without damaging the lower layer (for example, the enzyme layer). For example, the membrane can be applied directly on the enzyme layer without affecting the enzyme. In a preferred embodiment, the dispersion can be cast directly on the enzyme layer.

  In the method according to the present invention, the dispersion is preferably attached to the lower layer by physical adhesion. Therefore, no mechanical adhesion including the above-mentioned drawbacks is necessary for depositing the film.

  If desired, the dispersion can be dried after application onto the lower layer. Essentially any drying method known in the art can be used.

  The following Examples 1 and 2 will specifically explain the present invention.

Example 1
In order to prepare a membrane dispersion for quantifying the glucose concentration in a liquid sample, the following components were mixed.

  The mixture was applied as a 20 μm thick layer on top of the enzyme layer of an OptiCCA disposable glucose sensor (Roche Diagnostics Corp.). After drying at room temperature, a sensor spot (4 mm diameter) overcoated with the membrane according to the invention was cut out from the sensor foil and placed in a flow-through cell. The channel was filled with the appropriate buffer before the sample was injected.

Next, the element strength generated by the luminescence quenching of the luminescent dye contained in the oxygen sensitive layer was measured. The following reaction rate measurements (fluorescence intensity change: ΔI / sec) were obtained using three different glucose control solutions that were measured with a vapor pressure meter to achieve an oxygen partial pressure of 150 Torr.

Example 2
Example 2 shows that sensor regeneration is rapid and that multiple measurements are possible (using a control solution containing 356 mg / dL glucose and an oxygen partial pressure of 150 torr). Measurement was performed 3 times). The sensor used in Example 2 was made according to Example 1, but was not completely identical to the sensor of Example 1 because the film was hand-painted.

Claims (20)

A diffusion membrane for use in an enzyme-based sensor,
(a) at least one polymeric material, and
(b) The diffusion membrane comprising pore-forming particles dispersed in the at least one polymer material.   The diffusion film according to claim 1, wherein the pore-forming particles have a size of 0.5 to 100 μm.   The diffusion membrane according to claim 1 or 2, wherein the pore-forming particles are present in an amount of 0.5 to 70% by weight.   The diffusion film according to claim 1, wherein the pore-forming particles include diatomaceous earth.   The diffusion film according to claim 1, wherein the film has a thickness of 0.5 to 1000 μm.   An enzyme-based sensor comprising the diffusion membrane according to claim 1. (i) at least one enzyme layer,
(ii) the cover layer, and
(iii) The enzyme-based sensor of claim 6, further comprising at least one lower layer.   The enzyme-based sensor according to claim 6 or 7, wherein the diffusion film is the cover layer.   The enzyme-based sensor according to any one of claims 6 to 8, wherein the diffusion film is directly coated on a lower layer.   10. The enzyme-based sensor according to any one of claims 6 to 9, wherein the diffusion film is attached to the lower layer by physical adhesion.   11. The enzyme-based sensor according to any one of claims 6 to 10, wherein the diffusion membrane is coated directly on the enzyme layer.   12. The enzyme-based sensor according to any one of claims 6 to 11, which is an optical sensor.   12. The enzyme-based sensor according to any one of claims 6 to 11, which is an electrochemical sensor.   Use of the enzyme-based sensor according to any one of claims 6 to 13 for the detection and / or quantification of enzyme substrates and / or co-substrates.   15. Use according to claim 14, wherein the enzyme substrate is glucose.   16. Use according to claim 14 or 15, wherein the quantification is performed on blood.   Use according to any one of claims 14 to 16, wherein multiple measurements are made. A method for producing an enzyme-based sensor comprising:
(i) (a) at least one polymer material, and
(b) forming a dispersion comprising pore-forming particles;
(ii) casting the dispersion directly on a lower layer to form a diffusion film;
(iii) optionally drying the dispersion.   The method of claim 18, wherein the dispersion is attached to the underlying layer by physical adhesion.   20. A method according to claim 18 or 19, wherein the dispersion is cast directly on the enzyme layer.